Characterization of intestinal O-glycome in reactive oxygen species deficiency

Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation resulting from an inappropriate inflammatory response to intestinal microbes in a genetically susceptible host. Reactive oxygen species (ROS) generated by NADPH oxidases (NOX) provide antimicrobial defense, redox signaling and gut barrier maintenance. NADPH oxidase mutations have been identified in IBD patients, and mucus layer disruption, a critical aspect in IBD pathogenesis, was connected to NOX inactivation. To gain insight into ROS-dependent modification of epithelial glycosylation the colonic and ileal mucin O-glycome of mice with genetic NOX inactivation (Cyba mutant) was analyzed. O-glycans were released from purified murine mucins and analyzed by hydrophilic interaction ultra-performance liquid chromatography in combination with exoglycosidase digestion and mass spectrometry. We identified five novel glycans in ileum and found minor changes in O-glycans in the colon and ileum of Cyba mutant mice. Changes included an increase in glycans with terminal HexNAc and in core 2 glycans with Fuc-Gal- on C3 branch, and a decrease in core 3 glycans in the colon, while the ileum showed increased sialylation and a decrease in sulfated glycans. Our data suggest that NADPH oxidase activity alters the intestinal mucin O-glycans that may contribute to intestinal dysbiosis and chronic inflammation.


Introduction
Inflammatory bowel disease (IBD), a chronic inflammatory disorder with rising prevalence worldwide, includes mainly Crohn's disease and ulcerative colitis [1].IBD is a multifactorial disease that may result from an inappropriate inflammatory response to intestinal microbes in a genetically susceptible host, with altered host-microbe interactions likely driving pathogenesis [2].Intimately involved in reciprocal signaling between microbe communities and host are epithelial NADPH oxidases (NOX/DUOX) that provide not only maintenance and restitution of the gut barrier, but also protection against pathogens.Several NADPH oxidases are expressed in colonocytes, intestinal stem cells and secretory cells where they maintain various biological functions by generating superoxide or hydrogen peroxide for redox signaling [3].Loss-of-function variants of NOX1, NOX2 and DUOX2 have been linked to pediatric IBD and CGD-IBD, respectively [4][5][6].Mice with single Nox1, Nox2 (Cybb) or Duox2 deletion or inactivation do not reflect human IBD pathogenesis, likely due to the multifactorial nature of colitis that cannot be mimicked in the protective standards of animal facilities [3].Susceptibility to barrier disruption and epithelial inflammation was achieved when providing a second hit (2-hit model), for example by crossing Nox2 and Nod2 knockout mice [7] or by establishing reactive oxygen species (ROS) deficiency in both the epithelial and the immune compartment by introducing a missense mutation in the NOX dimerization partner protein p22 phox (Cyba) [8].This Cyba mutation inactivates three Nox isoforms (Nox1-3), resulting in substantially decreased ROS generation, and mimics a mutational hot spot in chronic granulomatous disease (CGD) patients.Cyba nmf333 mice (Cyba mutant mice) were highly susceptible to severe colonic inflammation when challenged due to the underlying mucus, microbiota, and innate immune defects [8].
Glycosylation is the most common posttranslational modification of proteins.Changes in glycans were found in many diseases including chronic inflammatory diseases and cancer [9].Mucins are glycosylated proteins that constitute the main components of mucus on the surface of mucosal epithelial cells [10].In the intestine the predominant mucin is MUC2 which is Nand O-glycosylated, with O-glycosylation being far more abundant.Glycosylation permits water molecule incorporation for gel formation and restricts access of mammalian and bacterial proteases to the mucin protein core [11].Mucins have multiple roles within the intestinal mucosal barrier as they form a dense protective layer in the colon to prevent the entry of pathogenic organisms/substances, but also provide in the loose mucus layer glycosylated attachment sites for commensal bacteria, enabling colonization with a mutually beneficial mucusassociated microbiota [12].By maintaining the impenetrable dense mucus layer, mucin-type O-glycans protect from spontaneous colitis in mice [13].Intestinal inflammation is associated with changes in genes regulating epithelial glycosylation and alterations in synthesis, packing, storage and release of MUC2 mucin.Mucin analysis in IBD patients and mice challenged with colitis inducers revealed a simplified mucin O-glycome with truncated O-glycans and changes in terminal fucosylation, sialyation and sulfation [12,14,15].These alterations contribute to mucin degradation, increased barrier permeability, microbiota modification and inflammation [12,14].Fucosyltransferases (FUT) catalyze the transfer of L-fucose to various sugar acceptor substrates, and the resulting fucosylation plays an important role in signaling pathways, mucin modification and mucus-bacteria interactions.Triple α1,2 fucosyltransferase knockout mice (Fut1 -/-Fut2 -/-Sec1(Fut3) -/-mice) are completely deficient in H antigen on red blood cells.While mucin Muc2 and Fut1 expression were not altered in Cyba mutant mice, the expression of Fut2 was decreased by 30-40% [8].The α1,2-fucosyltransferase FUT2 catalyses the addition of fucose to terminal galactose, and in humans FUT2 nucleotide polymorphisms are associated with changes in the intestinal microbiota composition and predisposition to Crohn's disease [16].Genetic variation in FUT2 determines the ABH secretor status and in cooperation with the α-(1,3/4)-fucosyltransferase FUT3 the Lewis status.FUT3 variants are also considered an IBD risk factor, likely due to dysbiosis as fucosylated glycans serve as attachment sites and carbon source for microbiota.
To gain insight in how ROS deficiency may weaken the mucus barrier by altering the O-glycome, we characterized and compared the colonic and ileal mucin O-glycans in Cyba mutant mice with internal control mice.

Mice
B6 Tyr + -Cyba nmf333 /J mice (JAX#005445, [17]) were backcrossed to in-house bred C57BL/6J mice for more than 10 generations.Internal control Cyba +/+ (WT) mice were prepared by separating genotypes at the F2 generation, derived from F1 heterozygotes, and using separately maintained F3 and F4 generations for this study.All mice were housed in individually ventilated cages in the same room in an SPF facility operating at FELASA standards and were supplied with Teklad 2018 diet (Envigo) and sterile water ad libitum.All animal experiments were performed in accordance with EU Directive 86/609/EEC, approved by the UCD Ethics Committee and authorized by the Irish Regulatory Authorities.

Mucin purification
12-16-week-old [18] female and male Cyba nmf333 (Cyba mut) and internal Cyba +/+ (WT) control mice on C57BL/6J background were euthanized by cervical dislocation.Intestines were excised, colon and ileum horizontally cut and rinsed carefully with PBS.Colonic (whole colon) and ileal (terminal ileum 10 cm) mucins were isolated and purified as previously published [19].The procedure was modified by using 8M guanidine hydrochloride (Sigma) for the initial extraction procedure, and for desalting of the samples in the last step by using a Bio-Gel P-6D (Bio-Rad) column.
Microwave-assisted non-reductive β-elimination was performed as described in Wilkinson et al. [21].Briefly, the gels containing O-glycans were crushed, lyophilized, and resuspended in 250 μL 40% dimethylamine in water with 0.1 g/mL ammonium carbonate.Samples were incubated in a Monowave 450 microwave reactor at 600W (Anton Paar) under 12-minute microwave radiation at 70˚C.Samples were then neutralized with 1M HCl, desalted using a Hypersep Hypercarb SPE cartridge, lyophilized and fluorescently labelled with 2AB [22].The label excess was removed by a Hypersep Diol SPE cartridge [23].
Reductive β-elimination and desalting was performed according to Schulz et al [24] and modified for an in-gel release [25].

PGC-LC-MSn
Reductive β-eliminated O-glycan alditols were resuspended in 20 μL water and 10% was injected onto a liquid chromatography-electrospray ionization tandem mass spectrometer (LC-ESI-MS) on a 10 cm × 250 μm column packed in-house with 5 μm porous graphitized carbon particles (Thermo-Hypersil).The flow rate was 5 μL/min, and the column was kept at room temperature.Buffer A was 10 mM ammonium bicarbonate (ABC) and buffer B was 10 mM ABC in 80% acetonitrile.The gradient was 0-45% B (0-46 minutes), washing by 100% B (46-54 minutes), and equilibration with 0% B (54-78 minutes).The samples were analyzed in negative ion mode on a LTQ linear ion trap mass spectrometer (Thermo Electron), with an IonMax standard ESI source with a stainless-steel needle at -3.5 kV.The nebulizer gas was compressed air.The capillary was heated at 270˚C with voltage at -50 kV.Full scan (m/z 380-1800, two microscans, maximum ion time 100 ms, full AGC target of 30,000) was followed by data-dependent MS 2 or MS 3 scans (two microscans, maximum 100 ms, AGC target value of 10,000) with a normalized collision energy of 30%, isolation window of 2.5 units, activation q = 0.25, 30 ms).The MS signal threshold for MS 2 and MS 3 were 300 and 100 counts, respectively.Data acquisition was performed using Xcalibur 2.0.7 software (Thermo Scientific).MS interpretation was aided by comparison to published MS 2 spectra made available online on Unicarb DB (unicarb-db.expasy.org).

Results
Analysis of the colonic mucin O-glycosylation profile of ROS-deficient Cyba mutant mice will provide additional information on the relation between permanent decrease in intestinal ROS levels and mucin glycans.This study analyzed both colonic and ileal mucins as ROS generation was also strongly downregulated in the terminal ileum of Cyba mutant mice [8].Colonic and ileal mucins were purified from female and male Cyba nmf333 (Cyba mut) and internal C57BL/ 6J (WT) control mice.The yield for purified colonic mucin was 10.4 mg (WT, n = 9) and 13.3 mg (Cyba mut, n = 16) and for purified ileal mucin 4.1 mg (WT, n = 13) and 6.0 mg (Cyba mut, n = 21), respectively.As expected, the yield of ileal and colonic mucin was decreased in Cyba mutant mice.As the microbiota of Cyba mutant mice was dysbiotic, the expression of Fut3 was determined.Sec1 (Fut3) expression in colonic tissues of Cyba mutant and wildtype mice was comparable (Fig 1A).Detection of fucosylated oligosaccharides by staining of colonic tissues with Ulex europaeus agglutinin 1 (UEA-I) showed slightly less signal deep in the crypts of Cyba mutant mice, but the pronounced change in the thickness and density of MUC2 mucin between wildtype and mutant mice did not permit any quantitative assessment (Fig 1B ).
After extraction and purification of colonic and ileal mucins from WT and Cyba mut mice detailed O-glycan analyses were performed on the pooled WT and Cyba mut samples by fluorescence liquid chromatography (FLR-HILIC-UPLC) in combination with exoglycosidase digestions for quantification and mass spectrometry (PGC-LC-MS n ) for sequence characterization.This method shows good reproducibility with most chromatographic peaks with CV below 15-20% for mucins [21,27].However, in our sample performing ten replicates on pooled mucin glycan samples from colon and ileum, integrating 127 peaks, the reproducibility was found to be lower (S1 Table ), and therefore we focused on identifying glycan features.1A and 1B.Detailed glycan assignments using both methods are shown in S2 and S3 Tables.We used exoglycosidase enzyme arrays to identify specific linkages and digest most of the glycans to the cores.Namely, using sialidases ABS and NAN1 identified sialic acid linkages, galactosidases BTG and SPG galactoses linkages, fucosidases BKF and AMF fucose linkages, and GUH and JBH revealed the presence of N-Acetylglucosamine (GlcNAc) or N-Acetylgalactosamine (GalNAc) (S3 Table ).Exoglycosidase enzymes used, and their specificities are described in the Materials and Methods section.
When comparing colonic and ileal Cyba mut with WT samples, the same glycans were identified in both pools, namely core 1-4 glycans both fucosylated, sialylated and/or sulfated (Tables 1 and S2).Quantitative analyses were done using both PGC-LC-MS method and HILI-C-UPLC in combination with the exoglycosidase digestion method.PGC-LC-MS quantified glycans as % area from all identified glycans, and HILIC-UPLC similarly as % area from all identified glycans as well as % area from all released glycans.When comparing colonic and ileal Cyba mut with WT mucins, no major changes were found when taking into account all methods.In colon, there was an indication of a minor increase in glycans with terminal Hex-NAc, core 2 glycans with Fuc-Gal-on C3 branch and glycans containing (sialyl)Lewis x epitope, and a decrease in core 3 glycans ( Comparison of colonic and ileal glycans showed in WT and Cyba mut colon a pronounced increase of core 2, core 4, and fucosylated and sulfated glycans as well as a decrease in core 1 and sialylated glycans.This indicates differences in glycosylation in distinct areas of the murine intestine (Fig 5, S4 Table ), in line with previous publications on mouse ileum/colon glycosylation [30,31].

Discussion
Cyba mutant mice are highly susceptible to chemically induced colitis due to their compromised epithelial barrier and inability to mount an efficient microbicidal host response [8].At homeostasis the loss of superoxide generation by Nox1-3 resulted in a thin or absent dense mucus layer due to altered mucus secretion with direct contact of microbiota with the crypt epithelium and pronounced bacterial dysbiosis with increased abundance of mucus-associated Mucispirillum schaedleri.As the microbiota of Cyba mutant mice is dysbiotic, fucosyltransferase expression was measured.In the earlier study by Aviello and coworkers the expression of the fucosyltransferase Fut1 was not altered while expression of Fut2 was decreased by 30-40% [8].We found comparable Sec1 (Fut3) expression in colonic tissues of Cyba mutant and wildtype mice in this study (Fig 1A).Next, we performed comprehensive glycan analysis, consisting of a combination of liquid chromatography, exoglycosidase digestions and mass spectrometry which enabled us to characterize the glycans with more depth as previously published studies, especially in relation to linkages and specific residues which our method could separate.We found five additional novel glycans in the ileum ( Fig 3 and S2B Table).These methods are complementary and provide the most comprehensive glycan assignments by using recently developed microwave assisted nonreductive β-elimination for the HILIC preparation which improves reproducibility of O-glycan analyses [21].As we found slightly lower reproducibility in our glycan peaks, we focused on the comparison of glycan features rather than individual glycans.
The importance of the mucus layer in providing protection for the host's epithelial barrier and as niche for colonization and nutrition of commensal facultative anaerobe and oxygen tolerant bacteria prompted several mucin O-glycome studies in humans and mice to date.Mucin-type O-glycosylation in various segments of the colon and small intestine varies considerably, as expected by differential expression of glycosyltransferases, region-specific physicochemical changes in mucus layer structure and host physiology.Differences between humans and mice exist in the overall abundance of O-glycan core structures and in the prevalence of modifications such as fucosylation versus sialyation in the small intestine [31][32][33].In conventional C57BL/6N mice regiospecific distribution of core 2 and core 1 glycans correlates well with our analyses in conventional Cyba +/+ (WT) and Cyba mutant mice on a C57BL/6J background [30,31].
In colon core 1, 2, 3, and 4 glycans were detected, whereas in ileum core 1, 2 and 3(6) glycans were present (Figs 2 and 4, Tables 1 and S3).Comparison of ROS-deficient Cyba mutant mouse mucins with wildtype mouse mucins did not show pronounced differences in ileal glycan structures, although changes in mucus secretion or in situ preservation were evident by low mucin yields.When the individual glycans were pooled into the features, several differences between Cyba mutant and control mice were noted.Host ROS deficiency resulted in an increase in the levels of glycans with terminal HexNAc, core 2 glycans with Fuc-Gal-on C3 branch and glycans containing (sialyl)Lewis x epitope.A trend to decreased levels of core 3 glycans in Cyba mutant mouse colon (Fig 3, S4 Table ) was also observed.Further, when comparing ileal mucins in Cyba mutant with control mice, an increase in sialylated and a decrease in sulfated glycans was detected (Fig 3 , S4 Table).
Changes in mucin O-glycans have been reported in IBD patients and in mice in association with spontaneous or heightened susceptibility to colitis.In IBD patients MUC2 protein, but not mRNA, was found to be increased, hypoglycosylated and/or poorly sulfated [34].It was suggested that glycotransferases in the goblet cell secretory apparatus or mucin glycan degrading enzymes, either mammalian or bacterial, may be responsible for the distinct glycan profiles [35].MUC2 protein of patients with active UC was composed of smaller glycans than in UC remission or in non-IBD individuals, suggesting that altered glycosylation in UC is closely associated with clinical severity of disease and may return to normal during remission [35].Sialylation was increased in UC, suggesting that inflammation affects the glycosylation of mucins.
An increase in sialyl Lewis x epitope, upregulated in inflammation, was also observed in IBD in line with our findings [15].Indeed, the glycosylation changes observed in IBD were found to coincide with inflammation in acute infections [36].An upregulation of sialyltransferases results in an increase in the STn antigen (sialosyl-GalNAc-S/T) on MUC2, which leads to smaller glycans, less complex glycosylation and a subsequent decrease in mucin sulfation [35,37].Mice lacking the sulfate transporter NaS1 have reduced intestinal mucin sulfation and increased susceptibility to bacterial infection and DSS-induced colitis [38].Consistent with these findings, mice lacking N-acetylglucosamine-6-O-sulfotransferase, which catalyzes the transfer of sulfates to mucins, showed decreased mucin sulfation, increased inflammation and increased susceptibility to DSS-induced colitis [39].Mice lacking both intestinal core 1-and core 3-derived O-glycans developed spontaneous colitis in both distal and proximal colon [13].Absence of core 3 O-glycans in mice lacking the β1,3-N-acetylglucosaminyl-transferase enzyme resulted in decreased MUC2 synthesis and a diminished mucus barrier, leading to increased susceptibility to dextran sulfate sodium (DSS)-induced colitis and development of colorectal tumors [40].Similar results were reported in mice lacking the core 2 β 1,6-N-acetylglucosaminyl-transferase enzyme [41].While mucins have not been analyzed in pediatric IBD patients with decreased oxidase activity due to mutational NOX1, NOX2 complex or DUOX2 inactivation, we show here that mucins of Cyba mutant mice, a murine model for a subset of CGD patients, displayed more sialyation and decreased sulfation at homeostasis, which together with decreased mucus secretion reflects an inflammation-susceptible environment.
Deviations in post-translational modifications of mucins such as glycosylation and sulfation have an impact on mucin degradation [35,42,43].Modified mucins will select for particular bacterial species that can attach, colonize and thrive better in these conditions, thereby displacing other commensals and reducing microbiota diversity.For example, the increase in mucusdegrading bacteria in IBD such as Ruminococcus spp.may degrade the mucus layer and increase the direct exposure of colon epithelial cells to microbiota [44,45].In accord, the dysbiotic microbiota of mice with decreased ROS generation such as Cyba nmf333 mice or Ncf1 -/- mice (p47 phox knockout, i.e.Nox2 complex inactivation) harbored an abundance of mucusassociated Mucispirillum schaedleri or Akkermansia muciniphila [8,46].Mucin glycosylation is an important element of mucosal barrier function in the intestine, and genetically or environmentally-induced alteration of mucin glycosylation will increase susceptibility to IBD [14].

Conclusion
This is the first report describing O-glycans from mice with a disrupted intestinal barrier due to genetic ROS deficiency.Our combined methodology consisting of comprehensive O-glycan analysis of total mucin extract from mouse colon and ileum using HILIC-UPLC in combination with exoglycosidase enzyme digestions and MS enabled us to identify five novel glycans in ileum.Our results in Cyba nmf333 mice are consistent with reported O-glycan changes in intestinal mucins associated with colitis, expanding the insight into IBD pathogenesis.

Fig 2 .
Fig 2. Typical/pooled HILIC-UPLC profile of A) colonic and B) ileal O-glycans derived from WT and Cyba nmf333 mice.Main O-glycans are labelled.Detailed Oglycan composition of these samples is listed in Table1.

Fig 3 ,
S2A and S4 Tables).In contrast, in ileal Cyba mut structures an increase in sialylation and a decrease in sulfated glycans was indicated (Fig 4, S3A and S4 Tables).

Fig 4 .
Fig 4. Comparison of O-glycan features in wildtype (WT) and Cyba nmf333 mouse colon and ileum.Glycan features in A) colon and B) ileum that show differences in WT and Cyba nmf333 mice using all methods.Core 3 group in ileum contains a low amount of core 6 glycans.Graphs are based on quantitation by HILIC-UPLC method.https://doi.org/10.1371/journal.pone.0297292.g004

Table 1 .
A. List of all O-linked glycans from colonic mucins from WT and Cyba mut mice.B. List of all O-linked glycans from ileal mucins from WT and Cyba mut mice.